Confirmation of the stereochemistry of spiroviolene.

We confirm the previously revised stereochemistry of spiroviolene by X-ray crystallographically characterizing a hydrazone derivative of 9-oxospiroviolane, which is synthesized by hydroboration/oxidation of spiroviolene followed by oxidation of the resultant hydroxy group. An unexpected thermal boron migration occurred during the hydroboration process of spiroviolene that resulted in the production of a mixture of 1α-hydroxyspiroviolane, 9α- and 9ß-hydroxyspiroviolane after oxidation. The assertion of the cis-orientation of the 19- and 20-methyl groups provided further support for the revised cyclization mechanism of spiroviolene.

Crystal Structure Based Mutagenesis of Cattleyene Synthase Leads to the Generation of Rearranged Polycyclic Diterpenes.

The crystal structures of cattleyene synthase (apo-CyS), and CyS complexed with geranylgeranyl pyrophosphate (GGPP) were solved. The CySC59A variant exhibited an increased production of cattleyene and other diterpenes with diverse skeletons. Its structure showed a widened active site cavity explaining the relaxed selectivity. Isotopic labeling experiments revealed a remarkable cyclization mechanism involving several skeletal rearrangements for one of the novel diterpenes.

Pseudomonas zhaodongensis sp. nov., isolated from saline and alkaline soils.

Strain NEAU-ST5-21(T) was isolated from saline and alkaline soils in Zhaodong City, Heilongjiang Province, China. It was aerobic, Gram-stain-negative, rod-shaped and motile with a polar flagellum. It produced yellow-orange colonies with a smooth surface, and grew in the presence of 0-5 % (w/v) NaCl (optimum 0 %, w/v), at temperatures of 20-40 °C (optimum 28 °C) and at pH 7-11 (optimum pH 7). Phylogenetic analyses based on the separate 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD gene sequences indicated that strain NEAU-ST5-21(T) belongs to the genus Pseudomonas in the class Gammaproteobacteria. The most closely related species is Pseudomonas xanthomarina, whose type strain (KMM 1447(T)) showed gene sequence similarities of 99.0 % for 16S rRNA, 81.8 % for gyrB and 85.0 % for rpoD with strain NEAU-ST5-21(T). DNA-DNA hybridization values between strain NEAU-ST5-21(T) and P. xanthomarina DSM 18231(T), Pseudomonas kunmingensis CGMCC 1.12273(T), Pseudomonas stutzeri DSM 5190(T), Pseudomonas oleovorans subsp. lubricantis DSM 21016(T), Pseudomomas chengduensis CGMCC 2318(T), Pseudomonas alcaliphila DSM 17744(T) and Pseudomonas toyotomiensis DSM 26169(T) were 52±0 % to 25±2 %. The DNA G+C content of strain NEAU-ST5-21(T) was 65 mol%. The major fatty acids (>10 %) were C18 : 1ω7c and/or C18 : 1ω6c, C16 : 1ω7c and/or C16 : 1ω6c and C16 : 0, the predominant respiratory quinone was ubiquinone 9, and polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, one unknown phospholipid, phosphatidylglycerol, one unknown aminolipid, one unknown lipid and a glycolipid. The proposed name is Pseudomonas zhaodongensis sp. nov., NEAU-ST5-21(T) ( = ACCC 06362(T) = DSM 27559(T)) being the type strain.

Halobacillus andaensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soil.

A Gram-stain-positive, endospore-forming, moderately halophilic bacterial strain, NEAU-ST10-40T, was isolated from a saline and alkaline soil in Anda City, China. It was strictly aerobic, rod-shaped and motile by peritrichous flagella. It formed light yellow colonies and grew at NaCl concentrations of 3-15 % (w/v) (optimum, 8 %, w/v), at pH 7.0-9.0 (optimum, pH 8.0) and at 4-60 °C (optimum, 30 °C). It contained meso-diaminopimelic acid in the cell-wall peptidoglycan. Phylogenetic analysis based on 16S rRNA gene sequences indicated that it belonged to the genus Halobacillus. Levels of 16S rRNA gene sequence similarity between strain NEAU-ST10-40T and the type strains of related species of the genus Halobacillus ranged from 98.8 % (Halobacillus alkaliphilus FP5T) to 97.1 % (Halobacillus kuroshimensis IS-Hb7T). DNA-DNA hybridization relatedness values between strain NEAU-ST10-40T and H. alkaliphilus DSM 18525T, Halobacillus campisalis KCTC 13144T, Halobacillus yeomjeoni DSM 17110T, Halobacillus halophilus DSM 2266T, Halobacillus litoralis DSM 10405T, Halobacillus dabanensis DSM 18199T, Halobacillus salinus DSM 18897T, Halobacillus naozhouensis DSM 21183T, Halobacillus trueperi DSM 10404T and Halobacillus salsuginis DSM 21185T were from 43 ± 1 to 19 ± 1 % (mean ± sd). The DNA G+C content was 39.3 mol%. The major fatty acids (>10 %) were anteiso-C15:0, anteiso-C17:0 and iso-C16:0, the only respiratory quinone detected was MK-7, and polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown phospholipids and three unknown lipids. On the basis of the data presented, strain NEAU-ST10-40T is considered to represent a novel species, for which the name Halobacillus andaensis sp. nov. is proposed. The type strain is NEAU-ST10-40T ( = CGMCC 1.12153T = DSM 25866T).

Kocuria dechangensis sp. nov., an actinobacterium isolated from saline and alkaline soils.

A Gram-stain positive, strictly aerobic, non-motile and coccus-shaped actinobacterium, designated strain NEAU-ST5-33(T), was isolated from saline and alkaline soils in Dechang Township, Zhaodong City, PR China. It formed beige-yellow colonies and grew at NaCl concentrations of 0-5% (w/v) (optimum 0%), at pH 6.0-9.0 (optimum pH 7.0) and over a temperature range of 4-50 °C (optimum 35 °C). Based on 16S rRNA gene sequence analysis, strain NEAU-ST5-33(T) was phylogenetically closely related to the type strains of species of the genus Kocuria, Kocuria polaris CMS 76or(T), Kocuria rosea DSM 20447(T), Kocuria turfanensis HO-9042(T), Kocuria aegyptia YIM 70003(T), Kocuria himachalensis K07-05(T) and Kocuria flava HO-9041(T), with respective sequence similarities of 98.8%, 98.8%, 98.3%, 98.1%, 98.1% and 97.9%. DNA-DNA hybridization relatedness values of strain NEAU-ST5-33(T) with type strains of the closely related species ranged from 54 ± 1% to 34 ± 1%. The DNA G+C content was 61.2 mol%. The major fatty acids (>5%) were C15 : 0 anteiso, C15 : 0 iso and C16 : 1ω7c and/or C16 : 1ω6c. The major menaquinone detected was MK-8 (H2), and the polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unknown aminolipid and one unknown lipid. On the basis of the genotypic, chemotaxonomic and phenotypic data, we propose that strain NEAU-ST5-33(T) represents a novel species of the genus Kocuria, with the name Kocuria dechangensis sp. nov. The type strain is NEAU-ST5-33(T) ( = CGMCC 1.12187(T) = DSM 25872(T)).

Pseudomonas songnenensis sp. nov., isolated from saline and alkaline soils in Songnen Plain, China.

The strain NEAU-ST5-5(T) was isolated from the saline and alkaline soil in Songnen Plain, North East of China. The bacterium was found to be aerobic, Gram-stain negative, rod-shaped and motile by means of several polar flagella. It forms yellow-orange colonies with a radial wrinkled surface. Phylogenetic analyses based on the separate 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD gene sequences indicated that it belongs to the genus Pseudomonas in the class Gammaproteobacteria. Strain NEAU-ST5-5(T) shows gene sequence similarities of 98.8-97.1 % for 16S rRNA, 90.5-78.4 % for gyrB and 90.4-71.1 % for rpoD with type strains of the closely related species of the genus Pseudomonas, respectively. DNA-DNA hybridization relatedness between strain NEAU-ST5-5(T) and type strains of the most closely related species, Pseudomonas stutzeri DSM 5190(T), P. xanthomarina DSM 18231(T), P. kunmingensis CGMCC 1.12273(T), P. alcaliphila DSM 17744(T) and P. oleovorans subsp. lubricantis DSM 21016(T) were 43 ± 1 to 25 ± 2 %. The major fatty acids (>10 %) were determined to be C18:1 ω7c/C18:1 ω6c, C16:1 ω7c/C16:1 ω6c and C16:0, the predominant respiratory quinone was identified as ubiquinone 9 and polar lipids were found to consist of phosphatidylethanolamine, phosphatidylglycerol, diphosphatidylglycerol, one unknown phospholipid, one unidentified aminophospholipid and one unknown lipid. The genotypic, chemotaxonomic and phenotypic analysis indicated that strain NEAU-ST5-5(T) represents a novel species of the genus Pseudomonas, for which the name Pseudomonas songnenensis sp. nov. is proposed. The type strain is NEAU-ST5-5(T) (=ACCC 06361(T) = DSM 27560(T)).

Planococcus dechangensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soils in Dechang Township, Zhaodong City, China.

Strain NEAU-ST10-9(T) is a moderately halophilic, coccoid and non-motile bacterium isolated from saline and alkaline soils in the Dechang Township, Zhaodong City, China. The bacterium was found to be aerobic and Gram-stain positive. It forms orange colonies and grows at NaCl concentrations of 2-10 % (w/v) (optimum, 4 % w/v), at 4-50 °C (optimum, 30 °C) and at pH 6.0-10.0 (optimum, pH 7.0). Phylogenetic analyses based on 16S rRNA gene sequences indicated that it belongs to the genus Planococcus within the family Planococcaceae. The most closely related species was Planococcus maritimus, whose type strain (TF-9(T)) showed gene sequence similarities of 99.1 % for 16S rRNA, 83.7 % for gyrB and 87.0 % for rpoB with those of strain NEAU-ST10-9(T), respectively. DNA-DNA hybridization relatedness values between strain NEAU-ST10-9(T) and type strains P. maritimus DSM 17275(T) , P. rifietoensis DSM 15069(T) , P. plakortidis DSM 23997(T), P. citreus DSM 20549(T), P. maitriensis DSM 15305(T), P. salinarum KCTC 13584(T) and P. columbae DSM 17517(T) were from 55 ± 1 to 32 ± 2 %. The DNA G+C content was found to be 45.2 mol %. The major fatty acids (>5 %) were determined as C15:0 anteiso, C16:1 ω7c alcohol, C17:1 ω9c and C17:0 anteiso. The major menaquinones of strain NEAU-ST10-9(T) were identified as MK-7 and MK-8. The polar lipids were found to contain of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, phosphocholine and two unknown lipids. The genotypic, chemotaxonomic and phenotypic analysis indicated that strain NEAU-ST10-9(T) represents a novel species of the genus Planococcus, for which we proposed the name Planococcus dechangensis sp. nov. The type strain is NEAU-ST10-9(T) (=CGMCC 1.12151(T)=DSM 25871(T)).

Halomonas songnenensis sp. nov., a moderately halophilic bacterium isolated from saline and alkaline soils.

A moderately halophilic bacterium (strain NEAU-ST10-39T) was isolated from saline and alkaline soils in the oilfield of Daqing City, Heilongjiang Province, China. The strain was strictly aerobic, Gram-stain-negative, rod-shaped and motile by peritrichous flagella. Its colonies were yellow. It grew at NaCl concentrations of 0.2-15% (w/v) (optimum 4%, w/v), at temperatures of 4-40 °C (optimum 35 °C) and at pH 5-10 (optimum pH 7). It did not produce acids from sugars or alcohols. Its DNA G+C content was 57.4 mol%. Phylogenetic analyses based on 16S rRNA gene sequences and concatenated 16S rRNA, gyrB and rpoD gene sequences indicated that it belonged to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species were Halomonas axialensis, Halomonas meridiana and Halomonas aquamarina, whose types shared 98.3% (16S rRNA), 82.7% (gyrB) and 83.9-84.5% (rpoD) sequence similarity with strain NEAU-ST10-39T. The results of DNA-DNA hybridization assays showed 20±2%-50±1 % relatedness between strain NEAU-ST10-39T and the most closely related species including Halomonas axialensis DSM 15723T, Halomonas meridiana DSM 5425T, Halomonas aquamarina DSM 30161(T), Halomonas johnsoniae DSM 21197T, Halomonas stevensii DSM 21198T, Halomonas nanhaiensis CCTCC AB 2012911(T), Halomonas hamiltonii DSM 21196T and Halomonas arcis CGMCC 1.6494T. The major fatty acids were C18 : 1ω7c (47.2%), C16:1ω7c and/or C16:1ω6c (18.9%) and C16:0 (16.3%), the only respiratory quinone detected was ubiquinone 9 and polar lipids consisted of diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unknown phospholipids and three unknown lipids. The new isolate is proposed to represent a novel species with the name Halomonas songnenensis sp. nov., NEAU-ST10-39T (=CGMCC 1.12152T=DSM 25870T) being the type strain.

Halomonas zhaodongensis sp. nov., a slightly halophilic bacterium isolated from saline-alkaline soils in Zhaodong, China.

A slightly halophilic bacterium (strain NEAU-ST10-25(T)) was isolated from saline-alkaline soils in Zhaodong City, Heilongjiang Province, China. The strain is a Gram-negative, aerobic motile rod. It accumulates poly-ß-hydroxyalkanoate and produces exopolysaccharide. It produces beige-yellow colonies. Growth occurs at NaCl concentrations (w/v) of 0-15 % (optimum 3 %), at temperatures of 4-60 °C (optimum 35 °C) and at pH 6-12 (optimum pH 9). Its G+C content is 53.8 mol%. Phylogenetic analyses based on the separate 16S rRNA gene and concatenation of the 16S rRNA, gyrB and rpoD genes indicate that it belongs to the genus Halomonas in the class Gammaproteobacteria. The most phylogenetically related species is Halomonas alkaliphila DSM 16354(T), with which strain NEAU-ST10-25(T) showed 16S rRNA, gyrB and rpoD gene sequence similarities of 99.2, 82.3 and 88.2 %, respectively. The results of DNA-DNA hybridization assays showed 60.47 ± 0.69 % DNA relatedness between strain NEAU-ST10-25(T) and H. alkaliphila DSM 16354(T), 42.43 ± 0.37 % between strain NEAU-ST10-25(T) and Halomonas venusta DSM 4743(T) and 30.62 ± 0.43 % between strain NEAU-ST10-25(T) and Halomonas hydrothermalis DSM 15725(T). The major fatty acids are C18:1 ω7c (62.3 %), C16:0 (17.6 %), C16:1 ω7c/C16:1 ω6c (7.7 %), C14:0 (2.9 %), C12:0 3-OH (2.8 %), C10:0 (2.1 %) and C18:1 ω9c (1.6 %) and the predominant respiratory quinone is ubiquinone 9 (Q-9). The proposed name is Halomonas zhaodongensis, NEAU-ST10-25(T) (=CGMCC 1.12286(T) = DSM 25869(T)) being the type strain.